The research plan involves four major interrelated aspects of central cholinergic neurons. Cholinergic muscarinic receptors will be localized by light microscopic autoradiography of specific 3H-QNB binding sites in frozen, unfixed tissue sections. Our past work has focused on receptors in the telencephalon, and, in this project, the caudal brainstem will be the main area of study. Another aspect of this application deals with sodium-dependent high affinity choline uptake, a process that may be a rate-limiting, regulatory step in the synthesis of acetylcholine. The following areas, aimed at elucidating the mechanisms and significance of choline transport, will be examined: post-mortem and decay changes in the uptake; the relationship of releasable acetylcholine to this choline transport system; the mechanism by which the choline transport is coupled to neuronal activity; and radiolabelled ligand binding to the transport system. Another aspect of this grant aims at mapping the location of cholinergic nerve terminals in the rat brain, by the light microscopic autoradiographic localization of sodium-dependent choline uptake sites in tissue slices in vitro. This takes advantage of the fact that sodium-dependent high affinity uptake sites are highly or selectively localized to cholinergic neurons. The last section of the grant is a critical evaluation of whether cholinergic nerve terminals in the CNS have the so called presynaptic receptors, which presumably modulate neurotransmitter release. Experimental conditions will be rigorously selected and extended to test if drug effects on acetylcholine release in vitro have the characteristics of receptors located on nerve terminals.